Target identification of Mycobacterium tuberculosis phenotypic\textit{Mycobacterium tuberculosis phenotypic} hits using a concerted chemogenomic, biophysical and structural approach

Mycobacterium phenotypic hits are a good reservoir for new chemotypes for the treatment of tuberculosis. However, the absence of defined molecular targets and modes of action could lead to failure in drug development. Therefore, a combination of ligand-based and structure-based chemogenomic approaches followed by biophysical and biochemical validation have been used to identify targets for Mycobacterium tuberculosis phenotypic hits. Our approach identified EthR and InhA as targets for several hits, with some showing dual activity against these proteins. From the 35 predicted EthR inhibitors, eight exhibited an IC50 below 50 μM against M. tuberculosis EthR and three were confirmed to be also simultaneously active against InhA. Further hit validation was performed using X-ray crystallography yielding eight new crystal structures of EthR inhibitors. Although the EthR inhibitors attain their activity against M. tuberculosis by hitting yet undefined targets, these results provide new lead compounds that could be further developed to be used to potentiate the effect of EthA activated pro-drugs, such as ethionamide, thus enhancing their bactericidal effect.GM is grateful to the European Molecular Biology Laboratory and Marie Sklodowska-Curie Actions for funding this work. VM and MB acknowledge Bill & Melinda Gates Foundation [subcontract by the Foundation for the National Institutes of Health (NIH)] (OPP1024021). VM and MS acknowledge the European Community’s Seventh Framework Programme [grant number 260872]. GP would like to acknowledge the Wellcome Trust and the European Molecular Biology Laboratory for funding. JPO was funded by the member nation states of the European Molecular Biology Laboratory. TLB acknowledges The Wellcome Trust for funding and support (grant number 200814/Z/16/Z)

Inhibiting mycobacterial tryptophan synthase by targeting the inter-subunit interface

Drug discovery efforts against the pathogen Mycobacterium tuberculosis (Mtb) have been advanced through phenotypic screens of extensive compound libraries. Such a screen revealed sulfolane 1 and indoline-5-sulfonamides 2 and 3 as potent inhibitors of mycobacterial growth. Optimization in the sulfolane series led to compound 4, which has proven activity in an in vivo murine model of Mtb infection. Here we identify the target and mode of inhibition of these compounds based on whole genome sequencing of spontaneous resistant mutants, which identified mutations locating to the essential α- and β-subunits of tryptophan synthase. Over-expression studies confirmed tryptophan synthase as the biological target. Biochemical techniques probed the mechanism of inhibition, revealing the mutant enzyme complex incurs a fitness cost but does not prevent inhibitor binding. Mapping of the resistance conferring mutations onto a low-resolution crystal structure of Mtb tryptophan synthase showed they locate to the interface between the α- and β-subunits. The discovery of anti-tubercular agents inhibiting tryptophan synthase highlights the therapeutic potential of this enzyme and draws attention to the prospect of other amino acid biosynthetic pathways as future Mtb drug targets

THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria

Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors

Mycobacterial dihydrofolate reductase inhibitors identified using chemogenomic methods and in vitro validation.

The lack of success in target-based screening approaches to the discovery of antibacterial agents has led to reemergence of phenotypic screening as a successful approach of identifying bioactive, antibacterial compounds. A challenge though with this route is then to identify the molecular target(s) and mechanism of action of the hits. This target identification, or deorphanization step, is often essential in further optimization and validation studies. Direct experimental identification of the molecular target of a screening hit is often complex, precisely because the properties and specificity of the hit are not yet optimized against that target, and so many false positives are often obtained. An alternative is to use computational, predictive, approaches to hypothesize a mechanism of action, which can then be validated in a more directed and efficient manner. Specifically here we present experimental validation of an in silico prediction from a large-scale screen performed against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The two potent anti-tubercular compounds studied in this case, belonging to the tetrahydro-1,3,5-triazin-2-amine (THT) family, were predicted and confirmed to be an inhibitor of dihydrofolate reductase (DHFR), a known essential Mtb gene, and already clinically validated as a drug target. Given the large number of similar screening data sets shared amongst the community, this in vitro validation of these target predictions gives weight to computational approaches to establish the mechanism of action (MoA) of novel screening hit

Antimycobacterial drug discovery using Mycobacteria-infected amoebae identifies anti-infectives and new molecular targets

Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13-14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential "universal" targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK

Chronic Diseases in North-West Tanzania and Southern Uganda. Public Perceptions of Terminologies, Aetiologies, Symptoms and Preferred Management

Research outputs produced to support a quantitative population survey, quantitative health facility survey, focus groups and in-depth interviews performed by the projec

Letter from the African Independent Ethics Committee (AIEC): Advice for institutional ethics committees on process, roles, composition and decolonisation

The AIEC was set up to provide ethics advice to African researchers, initially with a bias on social work and development. Ultimately, the committee will be enlarged to include people who work with communities, African cultural experts, social scientists from other disciplines, professionals from other disciplines and lawyers. When fully fledged, the committee will be able to provide full ethics services. It was started by the African Social Work Network (ASWNet) to address a gap that exists currently in African research, that of inadequate ethics oversight. This letter will be published three times a year, once in each third of the year. The authors are the current members of the AIEC. In this first ethics letter, we provide a brief review of literature on African ethics, provide key definitions and advice to ethics on the process of developing committees, roles of stakeholders, composition and decolonising

Advances in schistosomiasis drug discovery based on natural products.

Schistosomiasis is a neglected tropical disease affecting over 250 million people worldwide. The disease is the second most prevalent neglected tropical disease after malaria. Treatment of schistosomiasis relies on the administration of praziquantel (also known as biltricide). Reliance on a single drug poses a threat to the public health system as the parasite may become resistant as shown by some laboratory findings. The possibility of the resistance rising to clinically significant levels has motivated the scientific community to search for new drug nominees. For a long time, natural products have always been a foundation for the identification of drug leads in the pharmaceutical industry. This paper reviews the progress made in the discovery of natural anti-schistosomal agents in the field of drug discovery. We focus mainly on natural products that have been tested on the schistosome parasite and exhibited potency. We also highlight applications of advanced techniques in drug discovery, with a major focus on computer-aided drug discovery methods. Specifically, we discuss structure-based drug discovery and ligand-based drug design approaches, with an emphasis on virtual screening

In silico Comparison of Antimycobacterial Natural Products with Known Antituberculosis Drugs

Caballero, J (Caballero, Julio). Univ Talca, Ctr Bioinformat & Simulac Mol, Fac Ingn, Talca, ChileThe chemical space based on physicochemical properties and structural features of a diverse group of natural products with reported in vitro activity against different Mycobacterium tuberculosis strains is investigated using in silico tools. This is compared to the chemical space occupied by drugs currently recommended for the treatment of various forms of tuberculosis as well as compounds in preclinical and clinical development. Docking studies exploring possible binding affinities and modes of two main clusters of natural products on two different mycobacterial targets are also reported. Our docking results suggest that scytoscalarol, an antibacterial and antifungal guanidine-bearing sesterterpene, can inhibit arabinosyltransferase Mtb EmbC, and the beta-carboline alkaloids 8-hydroxymanzamine A and manzamine A can bind to the oxidoreductase of Mtb INHA On this basis, these products showing high binding affinities to the two targets in silico could be rationally selected for in vitro testing against these targets and/or semisynthetic modification

Natural products : a potential source of malaria transmission blocking drugs?

The ability to block human-to-mosquito and mosquito-to-human transmission of Plasmodium parasites is fundamental to accomplish the ambitious goal of malaria elimination. The WHO currently recommends only primaquine as a transmission-blocking drug but its use is severely restricted by toxicity in some populations. New, safe and clinically effective transmission-blocking drugs therefore need to be discovered. While natural products have been extensively investigated for the development of chemotherapeutic antimalarial agents, their potential use as transmission-blocking drugs is comparatively poorly explored. Here, we provide a comprehensive summary of the activities of natural products (and their derivatives) of plant and microbial origins against sexual stages of Plasmodium parasites and the Anopheles mosquito vector. We identify the prevailing challenges and opportunities and suggest how these can be mitigated and/or exploited in an endeavor to expedite transmission-blocking drug discovery efforts from natural products.The South African Research Chairs Initiative of the Department of Science and Technology, administered through the South African National Research Foundation and the NRF.http://www.mdpi.com/journal/pharmaceuticalsam2021BiochemistryChemistryGeneticsMicrobiology and Plant PathologyParaclinical Science